Embryonic stem cells, as cells which can be generated from all of tridermic-derived cells and tissues, are obtained from inner cell mass (ICM) of blastocyst formed after about 4-5 days from fertilization, and therefore involve ethical problems. Further, because of their pluripotency, they can be highly proliferated and variously differentiated, but have problems that they are hard to control and may be converted into cancer cells.
On the other hand, adult stem cells can be obtained from bone marrow, umbilical cord blood, adipose tissues, etc., and thus, may avoid any ethical problem. Furthermore, it has been demonstrated that although adult stem cells have a plasticity inferior to embryonic stem cells, they have a multipotency, which allows them to differentiate into various cells including endothelium, bone, muscle, nerve cells.
Mesenchymal stem cells are a kind of adult stem cells, i.e. stem cells having a multipotency, which allows them to differentiate into hematopoietic system cells as well as tissues including muscle, nerve, bone, adipose tissue, etc. In general, they can be obtained through isolation from bone marrow, and in addition, can be obtained from various areas in the adult body. Adult stem cells derived from bone marrow have disadvantages in that their plasticity and proliferation potency are much poor as compared to other stem cells and the procedures for extracting them are invasive. Therefore, recently umbilical cord blood has come into notice as the alternative source of adult stem cells which can be used instead of bone marrow. Although the characteristics of umbilical cord blood-derived stem cells are substantially similar to those of bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived stem cells have the characteristic properties which are superior to bone marrow-derived stem cells in view of the plasticity and proliferation potency.
Meanwhile, mesenchymal stem cells are adult stem cells characterized in that they are adhered and grown on the surface of cell culture plate, and have been demonstrated that they can be differentiated into adipocytes, chondrocyte, osteocytes, etc. under appropriate culture conditions (Erickson et al., Biochem. Biophys. Res. Commun., 290: 763, 2002; Halvorsen et al., Tissue Eng., 7:729, 2001; Wickham et al., Clin. Orthop. Relat. Res., 412:196, 2003; Dragoo et al., J. Bone Joint Surg., Br., 85:740, 2003). In addition, it has been reported that mesenchymal stem cells not only have an ability to differentiate into mesodermic cells including adipocytes, muscle cells, osteocytes, chondrocyte, etc., but also can be differentiated into non-mesodermic cells including pancreatic endocrine cells, liver cells, vascular endothelial cells and myocardial cells, and thus, an effort to use mesenchymal stem cells as a tool of cell therapeutic agents or gene therapeutic agents via self-transplantation has been actively made (Chung Jin Sup, J Korean Soc Transplant, 22:183-196, 2008).
At the present technical level, to use adult stem cells as the cell therapeutic agent it is required to standardize the culture conditions under which undifferentiated state can be maintained. In addition, since adult stem cells isolated from tissues are present in the mixed state of various kinds of cells, the technology capable of culturing homogeneous adult stem cells in a mass scale is also one of the problems sought to be solved.
In particular, the method for isolating adult stem cells from tissues or blood can generally include, for example, cell sorting utilizing antibodies for numerous surface antigens. However, said method has the limitation that the surface antigens for adult stem cells should be understood, and further, its use is greatly restricted by the problems that the common surface antigen (hereinafter, referred to as “marker”) for adult stem cells has not been known; and markers for adult stem cells are not variously developed, and further, even the known markers show different degrees of expression depending on the differentiation state; and particularly, the sorting equipment is expensive.
In order to obtain adult stem cells in a large quantity, while maintaining a multipotent property, in vitro culture technologies have generally utilized the characteristic feature that said cells adhere to the cell culture plate.
Said method is accomplished by removing mononuclear cells from bone marrow or blood with density gradient centrifugation using Ficoll-Pague, and selectively culturing the adult stem cells adhered to the culture plate in the serum-containing culture solution. The cells obtained from the above procedures comprise adult stem cells, which may possibly be mixed with other mononuclear cells and other stem cells. However, such mixed cell culture conditions may result in differing the distribution degree of nutrients, thereby leading to the heterogeneity of cell differentiation state. In the end, the problem that said cells cannot be produced as the homogeneous cell population serves as the fatal disadvantage that when they are used as the therapeutic agent, the actual effect may be different from the intended effect. Therefore, there is an urgent need for development of the effective culture technology which can provide homogeneous adult stem cells in a large quantity.